Mining Mystery: The effect of

acid mining (sulfur compounds) on Bacillus Mycoides found on the stalk of wild rice

(Zizania palustris)

Cassandra Roy Grade 12

•  Wild Rice, Manoomin, is a vital part of the Anishinaabe diet –  Ojibwee people have

harvested wild rice (Zizania palustris) for over 2,000 years

•  But Ojibwe people are now concerned for their wild rice beds http://o.aolcdn.com/photo-hub/news_gallery/

7/1/711797/1302866984619.JPEG

Introduction

Introduction •  The largest deposit of copper

and nickel has been discovered in northern Minnesota –  Copper and nickel are

extracted from tons of sulfur-bearing rock

–  Creating sulfuric acid •  Leaches heavy metals

–  Mercury and arsenic –  Ground water, rivers, and

lakes –  Where wild rice beds may

be located (Kraker, 2012; Marcotty, 2011)

Introduction •  Minnesota Department of

Natural Resources analysis –  Natural wild rice stands –  Sulfate levels in 2000

Minnesota bodies of water –  Over thirty years

•  Predominantly –  Sulfate levels above 10 mg/

L –  No natural and self-

perpetuating wild rice stands exists in Minnesota (Maccabee, 2011)

http://www.nature.org/ourinitiatives/habitats/riverslakes/explore/explore-the-mississippi-river-day-1-afternoon-mother-natures-sacred-gift.xml

Question/Hypothesis

•  What is the effect of acid mining (sulfur compounds) on Bacillus mycoides found on the stock of wild rice stalk (Zizania palustris)?

•  If Bacillus mycoides cultured from wild rice stalk (Zizania palustris) is exposed to acid mine drainage (sulfur compounds), then colony growth will be negatively affected.

Method Table

Nutrient Agar (only SO4) Serial dilutions of 0, 10, 50, 100, and 200 mg/L of sulfate (S04).

•  Bacillus mycoides was inoculated on nutrient agar plates –  Three plates per concentration –  Twenty one trials –  Three control plates not inoculated

with bacteria and three without sulfate

–  Incubated at room temperature for three days

Nutrient Agar (only SO4)

•  Placed under a quadrant counter –  Percentage of bacteria

per plate was recorded

•  As the amount of sulfate increased the percentage of bacteria increased

Plain Agar (SO4) Effects of just sulfur compounds minus the nutrient affect •  Serial dilutions 10, 100 and 300 mg/

L (SO4) •  Sixty agar plates were inoculated

with Bacillus mycoides •  No growth occurred after three days

of incubation at room temperature •  Possibly the Bacillus mycoides culture

was dead.

The above procedure was again repeated with sixty agar plates, again, no growth occurred

Plain Agar With Wild Rice Stalks

•  Five plain agar plates •  Five grams (two

centimeters) of a sterile wild rice stalk

•  Inoculated with Bacillus mycoides

•  The agar plates did not visually show any growth

•  Minimal growth of Bacillus mycoides was indicated microscopically.

Agar 5% Glucose (Carbon Energy SO4, SO3, and S2 ) •  10, 100 and 300 mg/L)

of the three sulfur compounds

•  Plain agar and five percent glucose (carbon energy)

•  Sixty agar plates were inoculated

•  After a three day incubation, again no growth occurred

Agar 5% nutrient (Never Jelled)

•  Previous procedure was repeated –  Five percent nutrient, and

only nutrient agar –  Added to 100 milliliters of

sterile water

•  Growth was apparent, but the agar did not gel and bacterial growth could not be measured

95% Agar, 5% Nutrient Agar

•  Previous procedure was repeated –  95% plain agar with

5% nutrient agar •  Finally, measureable

growth occurred •  Sixty plates was held up to

a light •  Maximum growth was

measured –  Across the agar plates

•  The amount of bacterial growth per treatment was compared for statistical difference using a computer program called SPSS

•  A Univariate Analysis of Variance (ANOVA) was done to determine if there was a difference in bacterial colony growth due to the sulfur compounds or just due to chance. A probability or p value of p<.05 was used to determine significance.

Statistics

Conclusion •  The original hypothesis was if

Bacillus mycoides cultured from wild rice stalk (Zizania palustris) was exposed to acid mine drainage (sulfur compounds), then colony growth will be negatively affected

•  The hypothesis was partially supported depending on the nutrient situation

•  As the sulfur compound concentrations increased

–  Increased in high nutrient –  Bacterial growth significantly

decreased (p<0.017) low nutrients

•  Not for the 10 mg/L sulfur compound concentrations (p<0.301)

http://thetyee.ca/News/2011/05/23/MiningMess/

Conclusion •  Minnesota (1973) water quality

ruling –  Limiting sulfates to 10

milligrams per liter (mg/L) in wild rice waters.

(Maccabee, 2011)

•  Bacillus mycoides cultured from wild rice stalks –  Similar growth rates –  Significantly reduced growth

above 10 mg/L in wild rice waters

•  There appears to be a relationship between Sulfur compound levels, Bacillus mycoides growth and that of Wild Rice in Minnesota Lakes

•  Supporting the idea that B. mycoides is the unknown bacterial in Pastor, Walker, and Dewey (2011)

•  Pastor, Walker, and Dewey (2011) found four year cycling of wild rice stand growth

•  B. mycoides could be a key player in the cycling

•  In the presence of a unlimited nutrients like

–  Large biomass wild rice/Nutrient agar plate

–  The growth of B. mycoides increases

–  Breaking down wild rice liter –  High carbon to nitrogen ratio –  Diverting nitrogen from the

water

Conclusion

C:N

•  Yet when in a low nutrient agar –  Sulfur compounds appeared to have a

negative affect on bacterial growth. –  Subsequent low nitrogen levels leads to

wild rice biomass oscillations

C:N

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Acknowledgements

•  I would like to thank: •  Dr. Cynthia Welsh for six years of support

and mentoring •  Michael Gillespie biochemistry professor

at Fond du Lac Tribal College for his supervision and the use of his lab

•  Holly Pellerin for supporting my project and travels